The T2G mutant is a good inhibitor of MMP-9

As discussed above, residue 2 of TIMP interacts with the

S19 pocket of MMPs (Gomis-Ruth et al. 1997; FernandezCatalan et al. 1998; Iyer et al.
2007; Maskos et al. 2007)
and has a major influence on affinity for different MMPs.
Substitution of Thr2 by glycine, which has no side chain,
increases the Ki value of the N-TIMP-1 and -3 for MMP-
1, MMP-2, MMP-3, and MT1-MMP by at least two to
three orders of magnitude (Table 1; Meng et al. 1999; Wei
et al. 2005). However, the same mutation has small
effects on the inhibitory activity of N-TIMP-3 for
ADAM17/TACE (Wei et al. 2005). Here, we find that
this mutation in TIMP-1 has a much smaller ;20-fold
effect on the inhibition of MMP-7 and MMP-9 (Table 1).
In contrast to N-TIMP-1 and other mutants described
above, which have similar Ki values for MMP-2 and
MMP-9 (Table 1), the T2G mutant has a >2500-fold
higher affinity for MMP-9 relative to MMP-2, showing
that TIMP-1 can be engineered to select between the two
gelatinases. Similar results were obtained with N-TIMP-3
(data not shown), suggesting that the T2G mutation may
have similar effects on activity in all TIMPs. Combination with the AB2 mutation did not
improve the selectivity, since the AB2 loop generally increased the affinity
for those enzymes that are poorly inhibited by the T2G
mutant but not on the inhibition of MMP-7 or MMP-9 A comparison of the Ki values for
mutants with the
AB2 loop substitution plus other mutations with those
calculated by adding together their effects on the free
energy of binding (Table 1) shows that some combinations have synergistic rather than
additive effects. Most
notably, the AB2 loop mitigates the effect of the T2G and
T2R mutation on binding to certain MMPs. In the case
of the T2G mutant this compensation is particularly
strong for MMP-1, -2, and -3 inhibition while, for
the T2R mutant, MMP-3, MMP-7, and MMP-9 are most
affected.